Conformational flexibility of an antibody combining site composed of two identical V regions"
The variable portion of the light chain (VL) of protein 3 15 was studied as a model for antibody composed of two "like" chains. VL exists as a dimer which contains the binding subsite for the dinitrophenyl (Dnp) ring of Dnp ligands. Circular dichroism analysis of hapten-protein complex demonstrated that the geometry of the Dnp hapten in the ( V L ) ~ binding site is different from that in the Fv (composed of VL-VH) site. The VL dimer was found t o be present in two distinct, pH-dependent conformations which differ markedly in their binding properties, At pH 8.0 VL dimer binds two Dnp-lysine per dimer, whereas at pH 6.0 only one ligand is bound per ( V L ) ~. The association constant of ( V L ) ~ for Dnp-L-lysine at pH 6.0 is 3-4-fold higher than that a t pH 8.0. The geometry of the hapten in the combining site is also different in the two conformers of ( V L ) ~ as can be judged from the disappearance at pH 6 of the 480 nm and 390 nm absorbance bands in the difference spectrum of VL-bound Dnp-caproate. The pH-dependent changes in the difference spectra between bound and free ligands were used t o follow the transition between the two conformers of VL dimer. It was concluded that the pKa of this transition is at pH 6.9. The change in VL conformation is also expressed in a 2-3-fold enhancement of VL fluorescence between pH 6 and pH 8, suggesting that the conformation at pH 8 which binds two ligands is more open, whereas at pH 6 a more tight conformation exists, binding one ligand with higher affinity. Such changes were not observed with Fv which is composed of both VL and VH. These data suggest that an antibody site composed of two like chains (VL-VL) can undergo marked conformational changes at physiological pH values. The relevance of this finding t o problems of receptors o n thymus-derived lymphocytes is discussed.